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Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

A typical human cell expresses only a few thousand of the more than 30 000 genes contained within our chromosomes. The chromosomal infrastructure is essential for gene control, determining both active and repressed states. It is important not only to turn the right genes on but also to turn the right genes off. Histones and chromatin components have key roles in this decision making process. Mistakes have severe consequences. If as few as three inappropriate genes are turned off, a normal cell can be converted into a cancer cell. This epigenetic silencing of genes underlies a new approach to cancer therapy. Advances in the biochemistry and genetics of chromatin remodeling reveal that gene inactivation depends on the recruitment of enzymes that control the display of DNA within the chromosome. Mistargeting of these enzymes leads to tumorigenesis, but inhibition of their activity presents a novel approach to therapy.

The number of chromatin modifying and remodeling complexes implicated in genome control is growing faster than our understanding of the functional roles they play. We discuss recent in vitro experiments with biochemically defined chromatin templates that illuminate new aspects of action by histone acetyltransferases and ATP-dependent chromatin remodeling engines in facilitating transcription. We review a number of studies that present an 'ordered recruitment' view of transcriptional activation, according to which various complexes enter and exit their target promoter in a set sequence, and at specific times, such that action by one complex sets the stage for the arrival of the next one. A consensus emerging from all these experiments is that the joint action by several types of chromatin remodeling machines can lead to a more profound alteration of the infrastructure of chromatin over a target promoter than could be obtained by these enzymes acting independently. In addition, it appears that in specific cases one type of chromatin structure alteration (e.g., histone hyperacetylation) is contingent upon prior alterations of a different sort (i.e., ATP-dependent remodeling of histone-DNA contacts). The striking differences between the precise sequence of action by various cofactors observed in these studies may be - at least in part - due to differences between the specific promoters studied, and distinct requirements exhibited by specific loci for chromatin remodeling based on their pre-existing nucleoprotein architecture.

Transcriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. We used Xenopus oocytes to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v-ErbA, on partly and fully chromatinized TR-responsive templates in vivo. Repression by v-ErbA was not as efficient as that mediated by TR, was significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v-ErbA required mature chromatin to effect repression. We find that both v-ErbA and TR can recruit the corepressor N-CoR, but, in contrast to existing models, show a concomitant enrichment for HDAC3 that occurs without an association with Sin3, HDAC1/RPD3, Mi-2 or HDAC5. We propose a requirement for chromatin infrastructure in N-CoR/HDAC3-effected repression and suggest that the inability of v-ErbA to silence on partly chromatinized templates may stem from its impaired capacity to interfere with basal transcriptional machinery function. In support of this notion, we find v-ErbA to be less competent than TR for binding to TFIIB in vitro and in vivo.

We investigate the role of the transcriptional coactivator p300 in gene activation by thyroid hormone receptor (TR) on addition of ligand. The ligand-bound TR targets chromatin disruption independently of gene activation. Exogenous p300 facilitates transcription from a disrupted chromatin template, but does not itself disrupt chromatin in the presence or absence of ligand-bound receptor. Nevertheless, the acetyltransferase activity of p300 is required to facilitate transcription from a disrupted chromatin template. Expression of E1A prevents aspects of chromatin remodeling and transcriptional activation dependent on TR and p300. E1A selectively inhibits the acetylation of non-histone substrates. E1A does not prevent the assembly of a DNase I-hypersensitive site induced by TR, but does inhibit topological alterations and the loss of canonical nucleosome arrays dependent on the addition of ligand. Mutants of E1A incompetent for interaction with p300 partially inhibit chromatin disruption but still allow nuclear receptors to activate transcription. We conclude that p300 has no essential role in chromatin disruption, but makes use of acetyltransferase activity to stimulate transcription at a subsequent step.

Lineage specificity and temporal ordering of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In this report, we investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. We found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. We suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure.

We identify Xenopus NF-Y as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y-box sequences are required for the assembly of DNase I-hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. We clone Xenopus NF-YA, NF-YB and NF-YC and establish that NF-Y is the predominant Y-box-binding protein in Xenopus oocyte nuclei. NF-Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. We suggest two steps in chromatin modification at the Xenopus hsp70 promoter: first the binding of NF-Y to the Y-boxes to pre-set chromatin and second the recruitment of p300 to modulate transcriptional activity.

The pathways of synthesis and maturation of pre-messenger RNA in the nucleus have a direct effect on the translational efficiency of mRNA in the cytoplasm. The transcription of intron-less mRNA in vivo directs this mRNA towards translational silencing. The presence of an intron at the 5' end of the transcript relieves this silencing, whereas an intron at the 3' end further represses translation. These regulatory events are strongly dependent on the transcription of pre-mRNA in the nucleus. The impact of nuclear history on regulatory events in the cytoplasm provides a novel mechanism for the control of gene expression.

Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus TRbetaA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte nuclei, and eliminates regulation of transcription by TR-RXR. Expression of Xenopus RPD3p, the catalytic subunit of histone deacetylase, represses the TRbetaA promoter, but only after efficient assembly of the template into nucleosomes. In contrast, the unliganded TR-RXR represses templates only partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression by the deacetylase alone, compared with those needed in the presence of unliganded TR-RXR. In addition, whereas hormone-bound TR-RXR targets chromatin disruption as assayed through changes in minichromosome topology and loss of a regular nucleosomal ladder on micrococcal nuclease digestion, addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR-RXR can facilitate transcriptional repression in the absence of hormone through mechanisms in addition to recruitment of deacetylase, and disrupts chromatin structure through mechanisms in addition to the inhibition or release of deacetylase.

Coactivators and corepressors regulate transcription by controlling interactions between sequence-specific transcription factors, the basal transcriptional machinery and the chromatin environment. This review consider the access of nuclear and steroid receptors to chromatin, their use of corepressors and coactivators to modify chromatin structure and the implications for transcriptional control. The assembly of specific nucleoprotein architectures and targeted histone modification emerge as central controlling elements for gene expression.

The Xenopus thyroid hormone receptor betaA (TRbetaA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We determine the translational position of the nucleosome containing the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asymmetric protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 association is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRbetaA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRbetaA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome containing the TRE may have regulatory significance for expression of the TRbetaA gene.

In Xenopus, cells from the animal hemisphere are competent to form mesodermal tissues from the morula through to the blastula stage. Loss of mesodermal competence at early gastrula is programmed cell-autonomously, and occurs even in single cells at the appropriate stage. To determine the mechanism by which this occurs, we have been investigating a concomitant, global change in expression of H1 linker histone subtypes. H1 histones are usually considered to be general repressors of transcription, but in Xenopus they are increasingly thought to have selective functions in transcriptional regulation. Xenopus eggs and embryos at stages before the midblastula transition are deficient in histone H1 protein, but contain an oocyte-specific variant called histone B4 or H1M. After the midblastula transition, histone B4 is progressively substituted by three somatic histone H1 variants, and replacement is complete by early neurula. Here we report that accumulation of somatic H1 protein is rate limiting for the loss of mesodermal competence. This involves selective transcriptional silencing of regulatory genes required for mesodermal differentiation pathways, like muscle, by somatic, but not maternal, H1 protein.

Transcriptional repression occurs during meiotic maturation of Xenopus oocytes. Injection of a DNA template containing an hsp70 promoter into Xenopus oocytes, followed by progesterone-induced maturation has been used to demonstrate a dynamic competition between the assembly of transcription factor-containing nucleoprotein complexes and repressive nucleosomal arrays during the maturation process. In particular, it is shown that increased levels of injected heat shock protein, the transcriptional activator Gal4-VP16 or the DNA template itself all lead to reduced repression of transcription on maturation. Conversely, injection of additional histone increases repression. Repression of transcription is shown to be accompanied by the formation of a more regular array of nucleosomes and by an increase in the efficiency of nucleosome assembly on the injected plasmid. Meiotic maturation is therefore accompanied by replacement of transcription factor complexes by a repressive chromatin environment.

Chromatin disruption and transcriptional activation are both thyroid hormone-dependent processes regulated by the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). In the absence of hormone, TR-RXR binds to nucleosomal DNA, locally disrupts histone-DNA contacts and generates a DNase I-hypersensitive site. Chromatin-bound unliganded TR-RXR silences transcription of the Xenopus TRbetaA gene within a canonical nucleosomal array. On addition of hormone, the receptor directs the extensive further disruption of chromatin structure over several hundred base pairs of DNA and activates transcription. We define a domain of the TR protein necessary for directing this extensive hormone-dependent chromatin disruption. Particular TR-RXR heterodimers containing mutations in this domain are able to bind both hormone and their thyroid hormone receptor recognition element (TRE) within chromatin, yet are unable to direct the extensive hormone-dependent disruption of chromatin or to activate transcription. We distinguish the hormone-dependent disruption of chromatin and transcriptional activation as independently regulated events through the mutagenesis of basal promoter elements and by altering the position and number of TREs within the TRbetaA promoter. Chromatin disruption alone on a minichromosome is shown to be insufficient for transcriptional activation of the TRbetaA gene.

We demonstrate using a dinucleosome template that acetylation of the core histones enhances transcription by RNA polymerase III. This effect is not dependent on an increased mobility of the core histone octamer with respect to DNA sequence. When linker histone is subsequently bound, we find both a reduction in nucleosome mobility and a repression of transcription. These effects of linker histone binding are independent of core histone acetylation, indicating that core histone acetylation does not prevent linker histone binding and the concomitant transcriptional repression. These studies are complemented by the use of a Xenopus egg extract competent both for chromatin assembly on replicating DNA and for RNA polymerase III transcription. Incorporation of acetylated histones and lack of linker histones together facilitate transcription by >10-fold in this system; however, they have little independent effect on transcription. Thus core histone acetylation significantly facilitates transcription, but this effect is inhibited by the assembly of linker histones into chromatin.

The molecular mechanisms responsible for the remodeling of entire somatic erythrocyte nuclei in Xenopus laevis egg cytoplasm have been examined. These transitions in chromosomal composition are associated with the capacity to activate new patterns of gene expression and the re-acquisition of replication competence. Somatic linker histone variants H1 and H1 (0) are released from chromatin in egg cytoplasm, whereas the oocyte-specific linker histone B4 and HMG1 are efficiently incorporated into remodeled chromatin. Histone H1 (0) is released from chromatin preferentially in comparison with histone H1. Core histones H2A and H4 in the somatic nucleus are phosphorylated during this remodeling process. These transitions recapitulate the chromosomal environment found within the nuclei of the early Xenopus embryo. Phosphorylation of somatic linker histone variants is demonstrated not to direct their release from chromatin, nor does direct competition with cytoplasmic stores of linker histone B4 determine their release. However, the molecular chaperone nucleoplasmin does have an important role in the selective removal of linker histones from somatic nuclei. For Xenopus erythrocyte nuclei, this disruption of chromatin structure leads to activation of the 5S rRNA genes. These results provide a molecular explanation for the remodeling of chromatin in Xenopus egg cytoplasm and indicate the capacity of molecular chaperones to disrupt a natural chromosomal environment, thereby facilitating transcription.

We examined the structural and functional consequences of incorporating either histone H1, histone B4 or HMG1 into a synthetic dinucleosome containing two 5S rRNA genes. We found that all three proteins bind to linker DNA, stabilizing an additional 20 bp from micrococcal nuclease digestion and restrict nucleosome mobility. Histone H1 has the highest-affinity interaction with the dinucleosome; histone B4 and HMG1 associate with significantly reduced affinities. We found that histone H1 binds to the dinucleosome template with a dissociation constant (KD) of 7.4 nM, whereas the KD is 45 nM for histone B4 and 300 nM for HMG1. The KDs for the interaction of these proteins with naked DNA are 18 nM for H1, 80 nM for B4 and 300 nM for HMG1. The differences in association of these proteins with the dinucleosome are reflected in the efficiency with which the different proteins repress transcription from the 5S rRNA genes. Thus, although all three proteins can contribute to the organization of chromatin, the stability of the structures they assemble will vary. Our results provide a molecular explanation for the transcriptional promiscuity of Xenopus early embryonic chromatin, which is enriched in HMG1 and linker histone B4, but deficient in histone H1.

The high mobility group proteins 1 and 2 (HMG1/2) and histone B4 are major components of chromatin within the nuclei assembled during the incubation of Xenopus sperm chromatin in Xenopus egg extract. To investigate their potential structural and functional roles, we have cloned and expressed Xenopus HMG1 and histone B4. Purified histone B4 and HMG1 form stable complexes with nucleosomes including Xenopus 5S DNA. Both proteins associate with linker DNA and stabilize it against digestion with micrococcal nuclease, in a similar manner to histone H1. However, neither histone B4 nor HMG1 influence the DNase I or hydroxyl radical digestion of DNA within the nucleosome core. We suggest that HMG1/2 and histone B4 have a shared structural role in organizing linker DNA in the nucleosome.

Nucleosome mobility facilitates the transcription of chromatin templates containing only histone octamers. Inclusion of linker histones in chromatin inhibits nucleosome mobility, directs nucleosome positioning and represses transcription. Transcriptional repression by linker histone occurs preferentially on templates associated with histone octamers relative to naked DNA. Mobile nucleosomes and the restriction of mobility by linker histones might be expected to exert a major influence on the accessibility of chromatin to regulatory molecules.

We have examined the cause of transcriptional quiescence prior to the mid-blastula transition (MBT) in Xenopus laevis. We have found distinct requirements for transcription of class II and class III genes. An artificial increase of the amount of DNA present within the embryo over that found at the MBT allows precocious transcription of tRNA genes, but not of the adenovirus E4 or human cytomegalovirus (CMV) promoters. Thus titration of an inhibitor by exogenous DNA determines class III but not class II gene activation. We demonstrate that the action of the inhibitor depends on the association of core histones with DNA. The addition of exogenous TBP, together with an increase in the amount of DNA within the embryo, allows significant basal transcription of class II genes prior to the MBT, whereas it does not increase transcription of tRNA genes. To examine the activation of transcription above basal levels, we used a defined minimal promoter containing five Gal4 binding sites and the activator Gal4-VP16. Precocious transcriptional activation is directed by Gal4-VP16 prior to the MBT, demonstrating that a functional transcriptional machinery exists at this early developmental stage. Furthermore, since this activation can occur in the absence of exogenous TBP or chromatin titration, a transcription factor that can penetrate chromatin is sufficient for recruitment of this machinery to a promoter. Our results support the hypothesis that the temporal regulation of transcription during early embryogenesis in Xenopus reflects not only a titration of inhibitors by DNA, but also a deficiency in the activity of transcriptional activators prior to the MBT.

The role of chromosomal proteins HMG-14 and HMG-17 in the generation of transcriptionally active chromatin was studied in a Xenopus laevis egg extract which supports complementary DNA strand synthesis and chromatin assembly. Chromosomal proteins HMG-14/HMG-17 enhanced transcription from a chromatin template carrying a 5S rRNA gene, but not from a DNA template. The transcriptional potential of chromatin was enhanced only when these proteins were incorporated into the template during, but not after, chromatin assembly. HMG-14 and HMG-17 stimulate transcription by increasing the activity, and not the number, of transcribed templates. They unfold the chromatin template without affecting the nucleosomal repeat or decreasing the content of histone B4. We suggest that HMG-14/HMG-17 enhance transcription by inducing an extended conformation in the chromatin fiber, perhaps due to interactions with histone tails in nucleosomes. By disrupting the higher order chromatin structure HMG-14/HMG-17 increase the accessibility of target sequences to components of the transcriptional apparatus.

A cell-free system from Xenopus eggs was used to study the role of chromosomal protein HMG-17 in the generation of the chromatin structure of transcriptionally active genes. Addition of HMG-17 protein to the extracts, which do not contain structural homologs of the HMG-14/-17 protein family, indicates the protein is incorporated into the nascent template during replication, prior to completion of chromatin assembly. The protein binds to and stabilizes the structure of the nucleosomal core thereby improving the apparent periodicity of the nucleosomal spacing of nascent chromatin. Assembly of HMG-17 into the nascent chromatin structure significantly increased the transcription potential of the 5S RNA gene and satellite I chromatin. Kinetic studies indicate that the increase in transcriptional potential is observed only when HMG-17 is incorporated into nucleosomes during chromatin assembly.

We describe the transcriptional potentiation in estrogen responsive transcription extracts of the Xenopus vitellogenin B1 gene promoter through the formation of a positioned nucleosome. Nuclease digestion and hydroxyl radical cleavage indicate that strong, DNA sequence-directed positioning of a nucleosome occurs between -300 and -140 relative to the start site of transcription. Deletion of this DNA sequence abolishes the potentiation of transcription due to nucleosome assembly. The wrapping of DNA around the histone core of the nucleosome positioned between -300 and -140 creates a static loop in which distal estrogen receptor binding sites are brought close to proximal promoter elements. This might facilitate interactions between the trans-acting factors themselves and/or RNA polymerase. Such a nucleosome provides an example of how chromatin structure might have a positive effect on the transcription process.

Nucleosomes were assembled on a plasmid carrying a Xenopus somatic 5S RNA gene prepared at different superhelix densities. The gene was preferentially assembled into a positioned nucleosome which was stable to superhelical stress. No evidence for a conformational change in the nucleosome was found, even under extreme negative superhelical stress. Transcription in an extract from Xenopus oocyte nuclei was repressed to a degree which depended on the number of nucleosomes assembled. Topoisomerase activity in the extract was effectively inhibited by camptothecin, which had no effect on transcription. Transcription of reconstitutes remained repressed relative to naked plasmids, and was independent of superhelix density. Transcripts from reconstitutes were derived solely from nucleosome-free genes. Thus, a histone octamer positioned on the gene was sufficient to block its transcription. Tryptic removal of the core histone tail domains had no effect on transcription at any superhelix density. Transcription of reconstitutes containing H3/H4 tetramers was also repressed, but not eliminated (unlike reconstitutes containing octamers), and repression was independent of superhelix density. We suggest that removal of histones H2A/H2B from the nucleosome facilitates activation of transcription in the extract. We conclude that superhelical stress alone does not activate transcription of a 5S RNA gene assembled into a nucleosome in vitro.

We have used a Xenopus egg extract to show that a competition exists between the assembly of transcription complexes and nucleosomes on replicating 5S DNA. This competition results in the establishment of a transcriptionally repressed state for 5S DNA that is dependent on core histones but not on the precise positioning of the cores. The repression is selective, since satellite I DNA is not significantly repressed under these conditions. We demonstrate that the efficiency of chromatin assembly compared with transcription complex assembly is an important variable in determining gene activity.

We have investigated the effect of heat shock on primary cultures of male and female Xenopus laevis hepatocytes as a function of estrogen-induced vitellogenin gene expression. Coincident with the induction of heat-shock protein (hsp) synthesis, thermal stress abolishes the estrogen activated transcription and accumulation of vitellogenin mRNA, at the same time causing the destabilization of vitellogenin mRNA accumulated by prior treatment with the hormone. Exposure of the cells to estrogen before heat shock allows an immediate resumption of vitellogenin gene transcription on return to 26 degrees C. Heat shock applied to cells from hormonally naive male Xenopus extends the lag period preceding vitellogenin gene transcription upon return to normal temperatures. This transient and reversible paralysis of estrogen responsiveness is paralleled by reversible changes in the amount of nuclear estrogen receptor in the hepatocytes. Heat shock therefore offers a novel approach in the manipulation and analysis of the early stages of steroid hormonal regulation of gene expression.

The genome of Xenopus laevis contains two large families of class III genes (oocyte 5S RNA and satellite I DNA) that are repressed in somatic cells. Both gene families are actively transcribed in a soluble extract of X.laevis oocyte nuclei, using chromatin deficient in histone H1 as a template. The addition of histone H1, to this transcriptionally active chromatin, results in the dominant and selective repression of oocyte 5S RNA genes and satellite I DNA. Somatic 5S RNA genes remain active following histone H1 addition. Changes in chromatin structure could have a dominant role in the regulation of class III gene expression during Xenopus embryogenesis.

An extract of whole oocytes (oocyte S150) differentially transcribes Xenopus oocyte and somatic 5S RNA genes. In the oocyte S150, transcription complexes with different stabilities are assembled onto oocyte and somatic 5S DNA. The stability of the transcription complex is dependent on activities present in a fraction containing transcription factor TFIIIC. This fraction stabilizes the binding of the positive transcription factor TFIIIA to a somatic 5S RNA gene much more efficiently than it does to an oocyte gene. The oocyte S150 transcription extract is deficient in TFIIIC such that supplementation with a fraction enriched in this transcription factor selectively stimulates oocyte 5S DNA transcription. Previously it has been shown that an egg extract deficient in TFIIIA selectively transcribes somatic 5S RNA genes. Thus under conditions where there is differential stability of transcription complexes, limitation of either TFIIIA or TFIIIC may exaggerate the differential expression of two genes.